Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
The origin of the nervous system in all vertebrates can be traced to the end of gastrulation. At this time, the ectoderm in the dorsal side of the embryo changes its fate from epidermal to neural. The newly formed neuroectoderm thickens to form a flattened structure called the neural plate which is characterized, in some vertebrates, by a central groove (neural groove) and thickened lateral edges (neural folds). At its early stages of differentiation, the neural plate already exhibits signs of regional differentiation along its anterior posterior (A-P) and mediolateral axis (M-L). The neural folds eventually fuse at the dorsal midline to form the neural tube which will differentiate into brain at its anterior end and spinal cord at its posterior end. Closure of the neural tube creates dorsal/ventral differences by virtue of previous mediolateral differentiation. Thus, at the end of neurulation, the neural tube has a clear anterior-posterior (A-P), dorsal ventral (D-V) and mediolateral (M-L) polarities (see, for example, Principles in Neural Science (3rd), eds. Kandel, Schwartz and Jessell, Elsevier Science Publishing Company: NY, 1991; and Developmental Biology (3rd), ed. S. F. Gilbert, Sinauer Associates: Sunderland Mass., 1991). Inductive interactions that define the fate of cells within the neural tube establish the initial pattern of the embryonic vertebrate nervous system. In the spinal cord, the identify of cell types is controlled, in part, by signals from two midline cell groups, the notochord and floor plate, that induce neural plate cells to differentiate into floor plate, motor neurons, and other ventral neuronal types (van Straaten et al. (1988) Anat. Embryol. 177:317-324; Placzek et al. (1993) Development 117:205-218; Yamada et al. (1991) Cell 64:035-647; and Hatta et al. (1991) Nature 350:339-341). In addition, signals from the floor plate are responsible for the orientation and direction of commissural neuron outgrowth (Placzek, M. et al., (1990) Development 110: 19-30). Besides patterning the neural tube, the notochord and floorplate are also responsible for producing signals which control the patterning of the somites by inhibiting differentiation of dorsal somite derivatives in the ventral regions (Brand-Saberi, B. et al., (1993) Anat. Embryol. 188: 239-245; Porquie, O. et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5242-5246).
Another important signaling center exists in the posterior mesenchyme of developing limb buds, called the Zone of Polarizing Activity, or xe2x80x9cZPAxe2x80x9d. When tissue from the posterior region of the limb bud is grafted to the anterior border of a second limb bud, the resultant limb will develop with additional digits in a mirror-image sequence along the anteroposterior axis (Saunders and Gasseling, (1968) Epithelial-Mesenchymal Interaction, pp. 78-97). This finding has led to the model that the ZPA is responsible for normal anteroposterior patterning in the limb. The ZPA has been hypothesized to function by releasing a signal, termed a xe2x80x9cmorphogenxe2x80x9d, which forms a gradient across the early embryonic bud. According to this model, the fate of cells at different distances from the ZPA is determined by the local concentration of the morphogen, with specific thresholds of the morphogen inducing successive structures (Wolpert, (1969) Theor. Biol. 25:1-47). This is supported by the finding that the extent of digit duplication is proportional to the number of implanted ZPA cells (Tickle, (1981) Nature 254:199-202).
Although the existence of inductive signals in the ZPA has been known for years, the molecular identities of these signals are only now beginning to be elucidated. An important step forward has been the discovery that the secreted protein Sonic hedgehog (Shh) is produced in several tissues with organizing properties, including notochord, floor plate and ZPA (Echelard et al. (1993), Cell 75: 1417-1430; Bitgood, M. J. and A. P. McMahon (1995) Dev. Biol. 172:126-38). Misexpressing Shh mimics the inductive effects on ectopic notochord in the neural tube and somites (Echelard et al. (1993) supra) and also mimics ZPA function in the limb bud (Riddle et al. (1993) Cell 75:1401-16; Chang et al. (1994) Development 120: 3339-53).
The vertebrate family of hedgehog genes includes at least four members, e.g., paralogs of the single drosophila hedgehog gene. Exemplary hedgehog genes and proteins are described in PCT publications WO 95/18856 and WO 96/17924. Three of these members, herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds, and mammals. A fourth member, herein referred to as tiggie-winkle hedgehog (Thh), appears specific to fish. Desert hedgehog (Dhh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (Ihh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Shh, which as described above, is primarily involved in morphogenic and neuroinductive activities. Given the critical inductive roles of hedgehog polypeptides in the development and maintenance of vertebrate organs, the identification of hedghog interacting proteins is of paramount significance in both clinical and research contexts.
The present invention relates to the discovery of a new class of hedgehog-binding protein, referred to herein as HIP (for hedgehog interacting protein). The HIP polypeptides of the present invention include polypeptides which bind the products of the hedgehog gene family. Hedgehog family members are known for their broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.
In general, the invention features isolated HIP polypeptides, preferably substantially pure preparations of the subject HIP polypeptides. The invention also provides recombinantly produced HIP polypeptides. In preferred embodiments the polypeptide has a biological activity including the ability to bind a hedgehog protein with high affinity, e.g., with a nanomolar or smaller dissociation constant (KD). HIP polypeptides which specifically antagonize such activities, such as may be provided by truncation mutants, are also specifically contemplated.
In one embodiment, the polypeptide is identical with or homologous to a HIP polypeptide represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8, or the core polypeptide sequence thereof (e.g., corresponding to residues 16-678 of SEQ ID. 5 or 6). Related members of the HIP family are also contemplated, for instance, a HIP polypeptide preferably has an amino acid sequence at least 65%, 67%, 69%, 70%, 75% or 80% homologous to a polypeptide represented by SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 though polypeptides with higher sequence homologies of, for example, 82%, 85%, 90% and 95% or are also contemplated. In a preferred embodiment, the HIP polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid sequence represented in any one or more of SEQ ID. Nos: 1-4 and 9-14. Homologs of the subject HIP proteins also include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with a HIP ligand, e.g. a hedgehog polypeptide.
The HIP polypeptide can comprise a full length protein, such as represented in SEQ ID No: 5, SEQ ID No: 6 or SEQ ID No: 7, or it may include the core polypeptide sequence thereof (e.g., corresponding to residues 16-678 of SEQ ID. 5 or 6), or it can include a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length. In preferred embodiments, the HIP polypeptide includes a sufficient portion of the excellular ligand binding domain to be able to specifically bind to a hedgehog ligand, preferably with a KD of 9 xcexcM or less and even more preferably of 9 nM or less. Truncated forms of the protein include, but are not limited to, soluble ligand binding domain fragments.
In certain preferred embodiments, the invention features a purified or recombinant HIP polypeptide having a core polypeptide molecular weight of about 78.4 kd. In other embodiments, the peptide core of a mature HIP protein preferably has a molecular weight in the range of 38.6 to 76.8 kD. It will be understood that certain post-translational modifications, e.g., glycosylation, prenylation, myristylation and the like, can increase the apparent molecular weight of the HIP protein relative to the unmodified polypeptide chain.
The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the HIP protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the HIP polypeptide, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, e.g. the second polypeptide portion is an epitope tag.
In yet another embodiment, the invention features nucleic acids encoding HIP polypeptides, which have the ability to modulate, e.g., either mimic or antagonize, at least a portion of the activity of a wild-type HIP polypeptide. Exemplary HIP-encoding nucleic acid sequences are represented by SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4.
In another embodiment, the nucleic acids of the present invention include coding sequences which hybridize under stringent conditions with all or a portion of the coding sequences designated in one or more of SEQ ID Nos: 1-4. The coding sequences of the nucleic acids can comprise sequences which are identical to coding sequences represented in SEQ ID Nos: 1, 2, 3, 4, 9, 10, 11, 12, 13 or 14, or it can merely be homologous to those sequences. In preferred embodiments, the nucleic acids encode polypeptides which specifically modulate, by acting as either agonists or antagonists, one or more of the bioactivities of wild-type HIP polypeptides.
Furthermore, in certain preferred embodiments, the subject HIP nucleic acids will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the HIP gene sequences. Such regulatory sequences can be used in to render the HIP gene sequences suitable for use as an expression vector. The transcriptional regulatory sequence can be from a HIP gene, or from a heterologous gene.
This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing HIP proteins by employing said expression vectors.
In still other embodiments, the subject invention provides a gene activation construct, wherein the gene activation construct is deigned to recombine with a genomic HIP gene in a cell to provide, e.g., by heterologous recombination, a heterologous transcriptional regulatory sequence operatively linked to a coding sequence of a genomic HIP gene. Cells having genomic HIP genes modified by gene activation constructs are also specifically contemplated.
In yet another embodiment, the present invention provides nucleic acids which hybridize under stringent conditions to nucleic acid probes corresponding to at least 12 consecutive nucleotides of either sense or antisense sequences of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4; though preferably to at least 25 consecutive nucleotides; and more preferably to at least 40, 50 or 75 consecutive nucleotides of either sense or antisense sequence of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4.
Yet another aspect of the present invention concerns an immunogen comprising a HIP polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a HIP polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by one of SEQ ID No: 5, SEQ ID No: 6. SEQ ID No: 7 and/or SEQ ID No: 8.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the HIP immunogen.
The invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g., animals which include (and preferably express) a heterologous form of a HIP gene described herein, or which misexpress an endogenous HIP gene, e.g., an animal in which expression of one or more of the subject HIP proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed HIP alleles or for use in drug screening.
The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequences of any one or more of SEQ ID Nos: 1-4 and 9-14, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a HIP protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a HIP protein; e.g. measuring a HIP mRNA level in a cell, or determining whether a genomic HIP gene has been mutated or deleted. These so-called xe2x80x9cprobes/primersxe2x80x9d of the invention can also be used as a part of xe2x80x9cantisensexe2x80x9d therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject HIP proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, though primers of 25, 40, 50, or 75 nucleotides in length are also contemplated.
In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between a hedgehog protein and a HIP polypeptide receptor. An exemplary method includes the steps of (a) forming a reaction mixture including: (i) a hedgehog polypeptide, (ii) a HIP polypeptide, and (iii) a test compound; and (b) detecting interaction of the hedgehog and HIP polypeptides. A statistically significant change (potentiation or inhibition) in the interaction of the hedgehog and HIP polypeptides in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of hedgehog bioactivity for the test compound. The reaction mixture can be a cell-free protein preparation, e.g., a reconsistuted protein mixture or a cell lysate, or it can be a recombinant cell including a heterologous nucleic acid recombinantly expressing the HIP polypeptide.
In preferred embodiments, the step of detecting interaction of the hedgehog and HIP polypeptides is a competitive binding assay. In other preferred embodiments, the step of detecting interaction of the hedgehog and HIP polypeptides involves detecting, in a cell-based assay, change(s) in the level of an intracellular second messenger responsive to signaling mediated by the HIP polypeptide. In still another preferred embodiment, the step of detecting interaction of the hedgehog and HIP polypeptides comprises detecting, in a cell-based assay, change(s) in the level of expression of a gene controlled by a transcriptional regulatory sequence responsive to signaling by the HIP polypeptide.
In preferred embodiments, the steps of the assay are repeated for a variegated library of at least 100 different test compounds, more preferably at least 103, 104 or 105 different test compounds. The test compound can be, e.g., a peptide, a nucleic acid, a carbohydrate, a small organic molecule, or natural product extract (or fraction thereof).
The present invention further contemplates the pharmaceutical formulation of one or more agents identified in such drug screening assays.
In other embodiments, the present invention provides a molecule, preferably a small organic molecule, which binds to HIP and either mimics or antagonizes hedgehog-induced signaling in cells expressing HIP.
Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a cell by modulating HIP bioactivity, e.g., by potentiating or disrupting certain protein-protein interactions. In general, whether carried out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a HIP therapeutic so as to alter, relative to the cell in the absence of treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. Accordingly, the method can be carried out with HIP therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from a HIP protein or ligand binding of a HIP protein, e.g., a hedgehog protein. Other HIP therapeutics include antisense constructs for inhibiting expression of HIP proteins, dominant negative mutants of HIP proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of the wild-type HIP protein, and gene therapy constructs including gene activation constructs.
In one embodiment, the subject method of modulating HIP bioactivity can be used in the treatment of testicular cells, so as to modulate spermatogenesis. In another embodiment, the subject method is used to modulate osteogenesis, comprising the treatment of osteogenic cells with an agent that modulates HIP boactivity. Likewise, where the treated cell is a chondrogenic cell, the present method is used to modulate chondrogenesis. In still, another embodiment, the subject method can be used to modulate the differentiation of a neuronal cell, to maintain a neuronal cell in a differentiated state, and/or to enhance the survival of a neuronal cell, e.g., to prevent apoptosis or other forms of cell death. For instance the present method can be used to affect the differentiation of neuronal cells such as motor neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, and peptidergic neurons.
Another aspect of the present invention provides a method of determining if a subject, e.g. an animal patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation or apoptosis. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a HIP protein; or (ii) the mis-expression of a HIP gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a HIP gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble HIP protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a HIP gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the HIP gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the HIP gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a HIP protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the HIP protein.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring, Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.